Rapid, actionable diagnosis of urban epidemic leptospirosis using a pathogenic Leptospira lipL32-based real-time PCR assay

Abstract

Background: With a conservatively estimated 1 million cases of leptospirosis worldwide and a 5-10% fatality rate, the rapid diagnosis of leptospirosis leading to effective clinical and public health decision making is of high importance, and yet remains a challenge.

Methodology: Based on parallel, population-based studies in two leptospirosis-endemic regions in Brazil, a real-time PCR assay which detects lipL32, a gene specifically present in pathogenic Leptospira, was assessed for the diagnostic effectiveness and accuracy. Patients identified by active hospital-based surveillance in Salvador and Curitiba during large urban leptospirosis epidemics were tested. Real-time PCR reactions were performed with DNA-extracted samples obtained from 127 confirmed and 23 unconfirmed cases suspected of leptospirosis, 122 patients with an acute febrile illness other than leptospirosis, and 60 healthy blood donors.

Principal findings: The PCR assay had a limit of detection of 280 Leptospira genomic equivalents/mL. Sensitivity for confirmed cases was 61% for whole blood and 29% for serum samples. Sensitivity was higher (86%) for samples collected within the first 6 days after onset of illness compared to those collected after 7 days (34%). The real-time PCR assay was able to detect leptospiral DNA in blood from 56% of serological non-confirmed cases. The overall specificity of the assay was 99%.

Conclusions: These findings indicate that real-time PCR may be a reliable tool for early diagnosis of leptospirosis, which is decisive for clinical management of severe and life-threatening cases and for public health decision making.

Fig 1. Standards for Reporting of Diagnostic Accuracy (STARD) flow chart of the 150 suspected leptospirosis cases enrolled in the study.

Patients were selected in two Brazilian cities: Curitiba (A) and Salvador (B).

Fig 2. Effect of different matrices on the accuracy of the lipL32 real-time PCR test.

Microscopically counted leptospires were used to spike EWB, serum and ultrapure water to 10⁶ leptospires/mL. Samples were diluted ten-fold serially and the respective DNA extracts were quantified by real-time PCR. Each point represents the mean result of four spiking experiments performed on water, whole blood and serum. Error bars show the mean ± SD.

Fig 3. Positivity of the lipL32 real-time PCR assay according to days after onset of symptoms in suspected cases of leptospirosis.

The bars represent the percentage of positive whole blood samples stratified according to days with symptoms (error bars show the 95% CI). The dashed line shows the geometric mean of the reciprocal MAT titer, in accordance with days with symptoms.